RNAi modulation of RSV and therapeutic uses thereof

ABSTRACT

The present invention is based on the in vivo demonstration that RSV can be inhibited through intranasal administration of iRNA agents as well as by parenteral administration of such agents. Further, it is shown that effective viral reduction can be achieved with more than one virus being treated concurrently. Based on these findings, the present invention provides general and specific compositions and methods that are useful in reducing RSV mRNA levels, RSV protein levels and viral titers in a subject, e.g., a mammal, such as a human. These findings can be applied to other respiratory viruses.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.11/411,291, filed on Apr. 26, 2006, now issued as U.S. Pat. No.7,517,865, which is a continuation-in-part of U.S. application Ser. No.11/326,956, filed Jan. 6, 2006, now issued as U.S. Pat. No. 7,507,809,which claims the benefit of U.S. Provisional Application No. 60/642,364,filed Jan. 7, 2005, and U.S. Provisional Application No. 60/659,828,filed Mar. 9, 2005.

TECHNICAL FIELD

The invention relates to the field of respiratory syncytial viral (RSV)therapy and compositions and methods for modulating viral replication,and more particularly to the down-regulation of a gene(s) of arespiratory syncytial virus by oligonucleotides via RNA interferencewhich are administered locally to the lungs and nasal passage viainhalation/intranasally or systemically via injection/intravenous.

BACKGROUND

By virtue of its natural function the respiratory tract is exposed to aslew of airborne pathogens that cause a variety of respiratory ailments.Viral infection of the respiratory tract is the most common cause ofinfantile hospitalization in the developed world with an estimated91,000 annual admissions in the US at a cost of $300 M. Humanrespiratory syncytial virus (RSV) and parainfluenza virus (PIV) are twomajor agents of respiratory illness; together, they infect the upper andlower respiratory tracts, leading to croup, pneumonia and bronchiolitis(Openshaw, P. J. M. Respir. Res. 3 (Suppl 1), S15-S20 (2002), Easton, A.J., et al., Clin. Microbiol. Rev. 17, 390-412 (2004)). RSV alone infectsup to 65% of all babies within the first year of life, and essentiallyall within the first 2 years. It is a significant cause of morbidity andmortality in the elderly as well. Immunity after RSV infection isneither complete nor lasting, and therefore, repeated infections occurin all age groups. Infants experiencing RSV bronchiolitis are morelikely to develop wheezing and asthma later in life. Research foreffective treatment and vaccine against RSV has been ongoing for nearlyfour decades with few successes (Openshaw, P. J. M. Respir. Res. 3(Suppl 1), S15-S20 (2002), Maggon, K. et al, Rev. Med. Virol. 14,149-168 (2004)). Currently, no vaccine is clinically approved for eitherRSV. Strains of both viruses also exist for nonhuman animals such as thecattle, goat, pig and sheep, causing loss to agriculture and the dairyand meat industry (Easton, A. J., et al., Clin. Microbiol. Rev. 17,390-412 (2004)).

Both RSV contain nonsegmented negative-strand RNA genomes and belong tothe Paramyxoviridae family. A number of features of these viruses havecontributed to the difficulties of prevention and therapy. The viralgenomes mutate at a high rate due to the lack of a replicationalproof-reading mechanism of the RNA genomes, presenting a significantchallenge in designing a reliable vaccine or antiviral (Sullender, W. M.Clin. Microbiol. Rev. 13, 1-15 (2000)). Promising inhibitors of the RSVfusion protein (F) were abandoned partly because the virus developedresistant mutations that were mapped to the F gene (Razinkov, V., et.al., Antivir. Res. 55, 189-200 (2002), Morton, C. J. et al. Virology311, 275-288 (2003)). Both viruses associate with cellular proteins,adding to the difficulty of obtaining cell-free viral material forvaccination (Burke, E., et al., Virology 252, 137-148 (1998), Burke, E.,et al., J. Virol. 74, 669-675 (2000), Gupta, S., et al., J. Virol. 72,2655-2662 (1998)). Finally, the immunology of both, and especially thatof RSV, is exquisitely complex (Peebles, R. S., Jr., et al., Viral.Immunol. 16, 25-34 (2003), Haynes, L. M., et al., J. Virol. 77,9831-9844 (2003)). Use of denatured RSV proteins as vaccines leads to“immunopotentiation” or vaccine-enhanced disease (Polack, F. P. et al.J. Exp. Med. 196, 859-865 (2002)). The overall problem is underscored bythe recent closure of a number of anti-RSV biopharma programs.

The RSV genome comprises a single strand of negative sense RNA that is15,222 nucleotides in length and yields eleven major proteins. (Falsey,A. R., and E. E. Walsh, 2000, Clinical Microbiological Reviews13:371-84.) Two of these proteins, the F (fusion) and G (attachment)glycoproteins, are the major surface proteins and the most important forinducing protective immunity. The SH (small hydrophobic) protein, the M(matrix) protein, and the M2 (22 kDa) protein are associated with theviral envelope but do not induce a protective immune response. The N(major nucleocapsid associated protein), P (phosphoprotein), and L(major polymerase protein) proteins are found associated with virionRNA. The two non-structural proteins, NS1 and NS2, presumablyparticipate in host-virus interaction but are not present in infectiousvirions.

Human RSV strains have been classified into two major groups, A and B.The G glycoprotein has been shown to be the most divergent among RSVproteins. Variability of the RSV G glycoprotein between and within thetwo RSV groups is believed to be important to the ability of RSV tocause yearly outbreaks of disease. The G glycoprotein comprises 289-299amino acids (depending on RSV strain), and has an intracellular,transmembrane, and highly glycosylated stalk structure of 90 kDa, aswell as heparin-binding domains. The glycoprotein exists in secreted andmembrane-bound forms.

Successful methods of treating RSV infection are currently unavailable(Maggon K and S. Barik, 2004, Reviews in Medical Virology 14:149-68).Infection of the lower respiratory tract with RSV is a self-limitingcondition in most cases. No definitive guidelines or criteria exist onhow to treat or when to admit or discharge infants and children with thedisease. Hypoxia, which can occur in association with RSV infection, canbe treated with oxygen via a nasal cannula. Mechanical ventilation forchildren with respiratory failure, shock, or recurrent apnea can lowermortality. Some physicians prescribe steroids. However, several studieshave shown that steroid therapy does not affect the clinical course ofinfants and children admitted to the hospital with bronchiolitis. Thuscorticosteroids, alone or in combination with bronchodilators, may beuseless in the management of bronchiolitis in otherwise healthyunventilated patients. In infants and children with underlyingcardiopulmonary diseases, such as bronchopulmonary dysphasia and asthma,steroids have also been used.

Ribavirin, a guanosine analogue with antiviral activity, has been usedto treat infants and children with RSV bronchiolitis since the mid1980s, but many studies evaluating its use have shown conflictingresults. In most centers, the use of ribavirin is now restricted toimmunocompromised patients and to those who are severely ill.

The severity of RSV bronchiolitis has been associated with low serumretinol concentrations, but trials in hospitalized children with RSVbronchiolitis have shown that vitamin A supplementation provides nobeneficial effect. Therapeutic trials of 1500 mg/kg intravenous RSVimmune globulin or 100 mg/kg inhaled immune globulin for RSVlower-respiratory-tract infection have also failed to show substantialbeneficial effects.

In developed countries, the treatment of RSV lower-respiratory-tractinfection is generally limited to symptomatic therapy. Antiviral therapyis usually limited to life-threatening situations due to its high costand to the lack of consensus on efficacy. In developing countries,oxygen is the main therapy (when available), and the only way to lowermortality is through prevention.

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.,Nature 391:806-811, 1998). Short dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function. RNAi has beensuggested as a method of developing a new class of therapeutic agents.However, to date, these have remained mostly as suggestions with nodemonstrate proof that RNAi can be used therapeutically.

Therefore, there is a need for safe and effective vaccines against RSV,especially for infants and children. There is also a need fortherapeutic agents and methods for treating RSV infection at all agesand in immuno-compromised individuals. There is also a need forscientific methods to characterize the protective immune response to RSVso that the pathogenesis of the disease can be studied, and screeningfor therapeutic agents and vaccines can be facilitated. The presentinvention overcomes previous shortcomings in the art by providingmethods and compositions effective for modulating or preventing RSVinfection. Specifically, the present invention advances the art byproviding iRNA agents that have been shown to reduce RSV levels in vitroand in vivo, as well as being effective against both major subtypes ofRSV, and a showing of therapeutic activity of this class of molecules.

SUMMARY

The present invention is based on the in vitro and in vivo demonstrationthat RSV can be inhibited through intranasal administration of iRNAagents, as well as by parenteral administration of such agents, and theidentification of potent iRNA agents from the P, N and L gene of RSVthat can reduce RNA levels with both the A and B subtype of RSV. Basedon these findings, the present invention provides specific compositionsand methods that are useful in reducing RSV mRNA levels, RSV proteinlevels and RSV viral titers in a subject, e.g., a mammal, such as ahuman.

In one aspect, the invention features an iRNA agent comprising twooligonucleotide sequences selected from those listed in Tables1(a)-1(c). In one embodiment, the iRNA agent comprises anoligonucleotide consisting of the sequence of SEQ ID NO:267, where theoligonucleotide is obtained by removing a silyl protecting group from anoligonucleotide precursor, where removal includes the steps of admixingthe oligonucleotide precursor of SEQ ID NO:267 with pyridine-HF,DMAP-HF, Urea-HF, TSAF, DAST, polyvinyl pyridine-HF or an aryl amine-HFreagent of formula AA:

where

-   R¹ is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;-   R² is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and-   R³ is aryl or heteroaryl.

In another aspect, the invention provides a method of making an isolatedoligonucleotide consisting of a sequence described in Tables 1(a)-1(c),where the method includes obtaining an oligonucleotide precursor bearinga silyl protecting group and admixing the oligonucleotide precursor withpyridine-HF, DMAP-HF, Urea-HF, TSAF, DAST, polyvinyl pyridine-HF or anaryl amine-HF reagent of formula AA:

where

-   R¹ is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;-   R² is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and-   R³ is aryl or heteroaryl    In one embodiment, the isolated oligonucleotide has the sequence of    SEQ ID NO:267.

The present invention also provides iRNA agents consisting of,consisting essentially of or comprising at least 15 or more contiguousnucleotides of one of the genes of RSV, particularly the P, N and Lgenes of RSV, and more particularly agents that comprising 15 or morecontiguous nucleotides from one of the sequence provided in Table1(a-c). The iRNA agent preferably consists of less than 30 nucleotidesper strand, e.g., 21-23 nucleotides, such as those provided in Tables1(a-c). The double stranded iRNA agent can either have blunt ends ormore preferably have overhangs of 1-4 nucleotides from one or both 3′ends of the agent.

Further, the iRNA agent can either contain only naturally occurringribonucleotide subunits, or can be synthesized so as to contain one ormore modifications to the sugar or base of one or more of theribonucleotide subunits that is included in the agent. The iRNA agentcan be further modified so as to be attached to a ligand that isselected to improve stability, distribution or cellular uptake of theagent, e.g. cholesterol. The iRNA agents can further be in isolated formor can be part of a pharmaceutical composition used for the methodsdescribed herein, particularly as a pharmaceutical compositionformulated for delivery to the lungs or nasal passage or formulated forparental administration. The pharmaceutical compositions can contain oneor more iRNA agents, and in some embodiments, will contain two or moreiRNA agents, each one directed to a different segment of a RSV gene orto two different RSV genes.

The present invention further provides methods for reducing the level ofRSV viral mRNA in a cell. Such methods comprise the step ofadministering one of the iRNA agents of the present invention to asubject as further described below. The present methods utilize thecellular mechanisms involved in RNA interference to selectively degradethe viral mRNA in a cell and are comprised of the step of contacting acell with one of the antiviral iRNA agents of the present invention.Such methods can be performed directly on a cell or can be performed ona mammalian subject by administering to a subject one of the iRNAagents/pharmaceutical compositions of the present invention. Reductionof viral mRNA in a cells results in a reduction in the amount of viralprotein produced, and in an organism, results in a decrease inreplicating viral titer (as shown in the Examples).

The methods and compositions of the invention, e.g., the methods andiRNA agent compositions can be used with any dosage and/or formulationdescribed herein, as well as with any route of administration describedherein. Particularly important is the showing herein of intranasaladministration of an iRNA agent and its ability to inhibit viralreplication in respiratory tissues.

One or both strands of strand an iRNA agent can be isolated according tomethods described in PCT publication No. WO2005/097817, which isincorporated herein by reference in its entirety.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thisdescription, the drawings, and from the claims. This applicationincorporates all cited references, patents, and patent applications byreferences in their entirety for all purposes.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: In vitro inhibition of RSV using iRNA agents. iRNA agentsprovided in Table 1(a-c) were tested for anti-RSV activity in a plaqueformation assay as described in the Examples. Each column (bar)represents an iRNA agent provided in Table 1(a-c), e.g. column 1 is thefirst agent in Table 1a etc. Active iRNA agents were identified.

FIG. 2: In vitro dose response inhibition of RSV using iRNA agents.Examples of active agents from Table 1 were tested for anti-RSV activityin a plaque formation assay as described in the Examples at fourconcentrations. A dose dependent response was found with active iRNAagent tested.

FIG. 3: In vitro inhibition of RSV B subtype using iRNA agents. iRNAagents provided in FIG. 2 were tested for anti-RSV activity againstsubtype B in a plaque formation assay as described in the Examples.Subtype B was inhibited by the iRNA agents tested.

FIG. 4: In vivo inhibition of RSV using iRNA agents. Agents as describedin the figure were tested for anti-RSV activity in a mouse model asdescribed in the Examples. The iRNA agents were effective at reducingviral titers in vivo.

FIG. 5: In vivo inhibition of RSV using AL-DP-1730. AL-DP-1730 wastested for dose dependent activity using the methods provided in theExamples. The agents showed a dose dependent response.

FIG. 6: In vivo inhibition of RSV using iRNA agents. iRNA agentsdescribed in the Figure were tested for anti-RSV activity in vivo asdescribed in the Examples.

FIG. 7: In vivo inhibition of RSV using iRNA agents. iRNA agentsdescribed in the Figure were tested for anti-RSV activity in vivo asdescribed in the Examples.

FIGS. 8A and 8B: In vivo inhibition of RSV using iRNA agents deliveredvia aerosol. iRNA agents described in the Figure were tested foranti-RSV activity in vivo as described in the Example.

FIG. 9: In vivo protection against RSV infection using iRNA agents. iRNAagents described in the Figure were tested prior to RSV challenge totest for protective activity.

FIG. 10: In vitro activity of nebulized iRNA agents. iRNA agents asdescribed were nebulized and shown to retain activity in an in vitroassay of RSV infection.

DETAILED DESCRIPTION

For ease of exposition the term “nucleotide” or “ribonucleotide” issometimes used herein in reference to one or more monomeric subunits ofan RNA agent. It will be understood that the usage of the term“ribonucleotide” or “nucleotide” herein can, in the case of a modifiedRNA or nucleotide surrogate, also refer to a modified nucleotide, orsurrogate replacement moiety, as further described below, at one or morepositions.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate, all of which are described herein or are wellknown in the RNA synthetic art. While numerous modified RNAs andnucleoside surrogates are described, preferred examples include thosewhich have greater resistance to nuclease degradation than do unmodifiedRNAs. Preferred examples include those that have a 2′ sugarmodification, a modification in a single strand overhang, preferably a3′ single strand overhang, or, particularly if single stranded, a5′-modification which includes one or more phosphate groups or one ormore analogs of a phosphate group.

An “iRNA agent” (abbreviation for “interfering RNA agent”) as usedherein, is an RNA agent, which can down-regulate the expression of atarget gene, e.g., RSV. While not wishing to be bound by theory, an iRNAagent may act by one or more of a number of mechanisms, includingpost-transcriptional cleavage of a target mRNA sometimes referred to inthe art as RNAi, or pre-transcriptional or pre-translational mechanisms.An iRNA agent can be a double stranded (ds) iRNA agent.

A “ds iRNA agent” (abbreviation for “double stranded iRNA agent”), asused herein, is an iRNA agent which includes more than one, andpreferably two, strands in which interchain hybridization can form aregion of duplex structure. A “strand” herein refers to a contiguoussequence of nucleotides (including non-naturally occurring or modifiednucleotides). The two or more strands may be, or each form a part of,separate molecules, or they may be covalently interconnected, e.g. by alinker, e.g. a polyethyleneglycol linker, to form but one molecule. Atleast one strand can include a region which is sufficientlycomplementary to a target RNA. Such strand is termed the “antisensestrand”. A second strand comprised in the dsRNA agent which comprises aregion complementary to the antisense strand is termed the “sensestrand”. However, a ds iRNA agent can also be formed from a single RNAmolecule which is, at least partly; self-complementary, forming, e.g., ahairpin or panhandle structure, including a duplex region. In such case,the term “strand” refers to one of the regions of the RNA molecule thatis complementary to another region of the same RNA molecule.

Although, in mammalian cells, long ds iRNA agents can induce theinterferon response which is frequently deleterious, short ds iRNAagents do not trigger the interferon response, at least not to an extentthat is deleterious to the cell and/or host. The iRNA agents of thepresent invention include molecules which are sufficiently short thatthey do not trigger a deleterious interferon response in mammaliancells. Thus, the administration of a composition of an iRNA agent (e.g.,formulated as described herein) to a mammalian cell can be used tosilence expression of an RSV gene while circumventing a deleteriousinterferon response. Molecules that are short enough that they do nottrigger a deleterious interferon response are termed siRNA agents orsiRNAs herein. “siRNA agent” or “siRNA” as used herein, refers to aniRNA agent, e.g., a ds iRNA agent, that is sufficiently short that itdoes not induce a deleterious interferon response in a human cell, e.g.,it has a duplexed region of less than 30 nucleotide pairs.

The isolated iRNA agents described herein, including ds iRNA agents andsiRNA agents, can mediate silencing of a gene, e.g., by RNA degradation.For convenience, such RNA is also referred to herein as the RNA to besilenced. Such a gene is also referred to as a target gene. Preferably,the RNA to be silenced is a gene product of an RSV gene, particularlythe P, N or L gene product.

As used herein, the phrase “mediates RNAi” refers to the ability of anagent to silence, in a sequence specific manner, a target gene.“Silencing a target gene” means the process whereby a cell containingand/or secreting a certain product of the target gene when not incontact with the agent, will contain and/or secret at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product whencontacted with the agent, as compared to a similar cell which has notbeen contacted with the agent. Such product of the target gene can, forexample, be a messenger RNA (mRNA), a protein, or a regulatory element.

In the anti viral uses of the present invention, silencing of a targetgene will result in a reduction in “viral titer” in the cell or in thesubject. As used herein, “reduction in viral titer” refers to a decreasein the number of viable virus produced by a cell or found in an organismundergoing the silencing of a viral target gene. Reduction in thecellular amount of virus produced will preferably lead to a decrease inthe amount of measurable virus produced in the tissues of a subjectundergoing treatment and a reduction in the severity of the symptoms ofthe viral infection. iRNA agents of the present invention are alsoreferred to as “antiviral iRNA agents”.

As used herein, a “RSV gene” refers to any one of the genes identifiedin the RSV virus genome (See Falsey, A. R., and E. E. Walsh, 2000,Clinical Microbiological Reviews 13:371-84). These genes are readilyknown in the art and include the N, P and L genes which are exemplifiedherein.

As used herein, the term “complementary” is used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between a compound of the invention and a target RNAmolecule, e.g. an RSV viral mRNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 4 nucleotides.

As used herein, an iRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA (e.g., a target RSV mRNA) if the iRNAagent reduces the production of a protein encoded by the target RNA in acell. The iRNA agent may also be “exactly complementary” to the targetRNA, e.g., the target RNA and the iRNA agent anneal, preferably to forma hybrid made exclusively of Watson-Crick base pairs in the region ofexact complementarity. A “sufficiently complementary” iRNA agent caninclude an internal region (e.g., of at least 10 nucleotides) that isexactly complementary to a target viral RNA. Moreover, in someembodiments, the iRNA agent specifically discriminates asingle-nucleotide difference. In this case, the iRNA agent only mediatesRNAi if exact complementarity is found in the region (e.g., within 7nucleotides of) the single-nucleotide difference. Preferred iRNA agentswill be based on or consist or comprise the sense and antisensesequences provided in the Examples.

As used herein, “essentially identical” when used referring to a firstnucleotide sequence in comparison to a second nucleotide sequence meansthat the first nucleotide sequence is identical to the second nucleotidesequence except for up to one, two or three nucleotide substitutions(e.g. adenosine replaced by uracil).

As used herein, a “subject” refers to a mammalian organism undergoingtreatment for a disorder mediated by viral expression, such as RSVinfection or undergoing treatment prophylactically to prevent viralinfection. The subject can be any mammal, such as a primate, cow, horse,mouse, rat, dog, pig, goat. In the preferred embodiment, the subject isa human.

As used herein, treating RSV infection refers to the amelioration of anybiological or pathological endpoints that 1) is mediated in part by thepresence of the virus in the subject and 2) whose outcome can beaffected by reducing the level of viral gene products present.

Design and Selection of iRNA Agents

The present invention is based on the demonstration of target genesilencing of a respiratory viral gene in vivo following localadministration to the lungs and nasal passage of an iRNA agent eithervia intranasal administration/inhalation or systemically/parenterallyvia injection and the resulting treatment of viral infection. Thepresent invention is further extended to the use of iRNA agents to morethan one respiratory virus and the treatment of both virus infectionswith co-administration of two or more iRNA agents.

Based on these results, the invention specifically provides an iRNAagent that can be used in treating viral infection, particularlyrespiratory viruses and in particular RSV infection, in isolated formand as a pharmaceutical composition described below. Such agents willinclude a sense strand having at least 15 or more contiguous nucleotidesthat are complementary to a viral gene and an antisense strand having atleast 15 or more contiguous nucleotides that are complementary to thesense strand sequence. Particularly useful are iRNA agents that consistof, consist essentially of or comprise a nucleotide sequence from the PN and L gene of RSV as provided in Table 1(a-c).

The iRNA agents of the present invention are based on and comprise atleast 15 or more contiguous nucleotides from one of the iRNA agentsshown to be active in Table 1(a-c). In such agents, the agent canconsist of consist essentially of or comprise the entire sequenceprovided in the table or can comprise 15 or more contiguous residuesprovided in Table 1a-c along with additional nucleotides from contiguousregions of the target gene.

An iRNA agent can be rationally designed based on sequence informationand desired characteristics and the information provided in Table1(a-c). For example, an iRNA agent can be designed according to sequenceof the agents provided in the Tables as well as in view of the entirecoding sequence of the target gene.

Accordingly, the present invention provides iRNA agents comprising asense strand and antisense strand each comprising a sequence of at least15, 16, 17, 18, 19, 20, 21 or 23 nucleotides which is essentiallyidentical to, as defined above, a portion of a gene from a respiratoryvirus, particularly the P, N or L protein genes of RSV. Exemplified iRNAagents include those that comprise 15 or more contiguous nucleotidesfrom one of the agents provided in Table 1(a-c).

The antisense strand of an iRNA agent should be equal to or at least,15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should beequal to or less than 50, 40, or 30, nucleotides in length. Preferredranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides inlength. Exemplified iRNA agents include those that comprise 15 or morenucleotides from one of the antisense strands of one of the agents inTable 1(a-c).

The sense strand of an iRNA agent should be equal to or at least 15, 1617, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should be equalto or less than 50, 40, or 30 nucleotides in length. Preferred rangesare 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.Exemplified iRNA agents include those that comprise 15 or morenucleotides from one of the sense strands of one of the agents in Table1(a-c).

The double stranded portion of an iRNA agent should be equal to or atleast, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 50nucleotide pairs in length. It should be equal to or less than 50, 40,or 30 nucleotides pairs in length. Preferred ranges are 15-30, 17 to 25,19 to 23, and 19 to 21 nucleotides pairs in length.

The agents provided in Table 1(a-c) are 21 nucleotide in length for eachstrand. The iRNA agents contain a 19 nucleotide double stranded regionwith a 2 nucleotide overhang on each of the 3′ ends of the agent. Theseagents can be modified as described herein to obtain equivalent agentscomprising at least a portion of these sequences (15 or more contiguousnucleotides) and or modifications to the oligonucleotide bases andlinkages.

Generally, the iRNA agents of the instant invention include a region ofsufficient complementarity to the viral gene, e.g. the P, N or L proteinof RSV, and are of sufficient length in terms of nucleotides, that theiRNA agent, or a fragment thereof, can mediate down regulation of thespecific viral gene. The antisense strands of the iRNA agents of thepresent invention are preferably fully complementary to the mRNAsequences of viral gene, as is herein for the P, L or N proteins of RSV.However, it is not necessary that there be perfect complementaritybetween the iRNA agent and the target, but the correspondence must besufficient to enable the iRNA agent, or a cleavage product thereof, todirect sequence specific silencing, e.g., by RNAi cleavage of an RSVmRNA.

Therefore, the iRNA agents of the instant invention include agentscomprising a sense strand and antisense strand each comprising asequence of at least 16, 17 or 18 nucleotides which is essentiallyidentical, as defined below, to one of the sequences of a viral gene,particularly the P, N or L protein of RSV, such as those agent providedin Table 1(a-c), except that not more than 1, 2 or 3 nucleotides perstrand, respectively, have been substituted by other nucleotides (e.g.adenosine replaced by uracil), while essentially retaining the abilityto inhibit RSV expression in cultured human cells, as defined below.These agents will therefore possess at least 15 or more nucleotidesidentical to one of the sequences of a viral gene, particularly the P, Lor N protein gene of RSV, but 1, 2 or 3 base mismatches with respect toeither the target viral mRNA sequence or between the sense and antisensestrand are introduced. Mismatches to the target viral mRNA sequence,particularly in the antisense strand, are most tolerated in the terminalregions and if present are preferably in a terminal region or regions,e.g., within 6, 5, 4, or 3 nucleotides of a 5′ and/or 3′ terminus, mostpreferably within 6, 5, 4, or 3 nucleotides of the 5′-terminus of thesense strand or the 3′-terminus of the antisense strand. The sensestrand need only be sufficiently complementary with the antisense strandto maintain the overall double stranded character of the molecule.

It is preferred that the sense and antisense strands be chosen such thatthe iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule, such as those exemplified in Table 1(a-c).Thus, an iRNA agent contains sense and antisense strands, preferablypaired to contain an overhang, e.g., one or two 5′ or 3′ overhangs butpreferably a 3′ overhang of 2-3 nucleotides. Most embodiments will havea 3′ overhang. Preferred siRNA agents will have single-strandedoverhangs, preferably 3′ overhangs, of 1 to 4, or preferably 2 or 3nucleotides, in length, on one or both ends of the iRNA agent. Theoverhangs can be the result of one strand being longer than the other,or the result of two strands of the same length being staggered. 5′-endsare preferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe siRNA agent range discussed above. Embodiments in which the twostrands of the siRNA agent are linked, e.g., covalently linked are alsoincluded. Hairpin, or other single strand structures which provide therequired double stranded region, and preferably a 3′ overhang are alsowithin the invention.

Evaluation of Candidate iRNA Agents

A candidate iRNA agent can be evaluated for its ability to down regulatetarget gene expression. For example, a candidate iRNA agent can beprovided, and contacted with a cell, e.g. a human cell, that has beeninfected with or will be infected with the virus of interest, e.g., avirus containing the target gene. Alternatively, the cell can betransfected with a construct from which a target viral gene isexpressed, thus preventing the need for a viral infectivity model. Thelevel of target gene expression prior to and following contact with thecandidate iRNA agent can be compared, e.g. on an RNA, protein level orviral titer. If it is determined that the amount of RNA, protein orvirus expressed from the target gene is lower following contact with theiRNA agent, then it can be concluded that the iRNA agent down-regulatestarget gene expression. The level of target viral RNA or viral proteinin the cell or viral titer in a cell or tissue can be determined by anymethod desired. For example, the level of target RNA can be determinedby Northern blot analysis, reverse transcription coupled with polymerasechain reaction (RT-PCR), bDNA analysis, or RNAse protection assay. Thelevel of protein can be determined, for example, by Western blotanalysis or immuno-fluorescence. Viral titer can be detected through aplaque formation assay.

Stability Testing, Modification, and Retesting of iRNA Agents

A candidate iRNA agent can be evaluated with respect to stability, e.g.,its susceptibility to cleavage by an endonuclease or exonuclease, suchas when the iRNA agent is introduced into the body of a subject. Methodscan be employed to identify sites that are susceptible to modification,particularly cleavage, e.g., cleavage by a component found in the bodyof a subject.

When sites susceptible to cleavage are identified, a further iRNA agentcan be designed and/or synthesized wherein the potential cleavage siteis made resistant to cleavage, e.g. by introduction of a 2′-modificationon the site of cleavage, e.g. a 2′-O-methyl group. This further iRNAagent can be retested for stability, and this process may be iterateduntil an iRNA agent is found exhibiting the desired stability.

In Vivo Testing

An iRNA agent identified as being capable of inhibiting viral geneexpression can be tested for functionality in vivo in an animal model(e.g., in a mammal, such as in mouse, rat or primate) as shown in theexamples. For example, the iRNA agent can be administered to an animal,and the iRNA agent evaluated with respect to its biodistribution,stability, and its ability to inhibit viral, e.g. RSV, gene expressionor reduce viral titer.

The iRNA agent can be administered directly to the target tissue, suchas by injection, or the iRNA agent can be administered to the animalmodel in the same manner that it would be administered to a human. Asshown herein, the agent can be preferably administered via inhalation asa means of treating viral infection.

The iRNA agent can also be evaluated for its intracellular distribution.The evaluation can include determining whether the iRNA agent was takenup into the cell. The evaluation can also include determining thestability (e.g., the half-life) of the iRNA agent. Evaluation of an iRNAagent in vivo can be facilitated by use of an iRNA agent conjugated to atraceable marker (e.g., a fluorescent marker such as fluorescein; aradioactive label, such as ³⁵S, ³²P, ³³P, or ³H; gold particles; orantigen particles for immunohistochemistry) or other suitable detectionmethod.

The iRNA agent can be evaluated with respect to its ability to downregulate viral gene expression. Levels of viral gene expression in vivocan be measured, for example, by in situ hybridization, or by theisolation of RNA from tissue prior to and following exposure to the iRNAagent. Where the animal needs to be sacrificed in order to harvest thetissue, an untreated control animal will serve for comparison. Targetviral mRNA can be detected by any desired method, including but notlimited to RT-PCR, Northern blot, branched-DNA assay, or RNAaseprotection assay. Alternatively, or additionally, viral gene expressioncan be monitored by performing Western blot analysis on tissue extractstreated with the iRNA agent or by ELISA. Viral titer can be determinedusing a pfu assay.

iRNA Chemistry

Described herein are isolated iRNA agents, e.g., ds RNA agents, thatmediate RNAi to inhibit expression of a viral gene, e.g. the P proteinof RSV.

RNA agents discussed herein include otherwise unmodified RNA as well asRNA which have been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, preferably as occur naturally in the human body. The art hasreferred to rare or unusual, but naturally occurring, RNAs as modifiedRNAs, see, e.g., Limbach et al., (1994) Nucleic Acids Res. 22:2183-2196. Such rare or unusual RNAs, often termed modified RNAs(apparently because these are typically the result of apost-transcriptional modification) are within the term unmodified RNA,as used herein. Modified RNA as used herein refers to a molecule inwhich one or more of the components of the nucleic acid, namely sugars,bases, and phosphate moieties, are different from that which occurs innature, preferably different from that which occurs in the human body.While they are referred to as modified “RNAs,” they will of course,because of the modification, include molecules which are not RNAs.Nucleoside surrogates are molecules in which the ribophosphate backboneis replaced with a non-ribophosphate construct that allows the bases tothe presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone. Examples of each of the above are discussed herein.

Modifications described herein can be incorporated into anydouble-stranded RNA and RNA-like molecule described herein, e.g., aniRNA agent. It may be desirable to modify one or both of the antisenseand sense strands of an iRNA agent. As nucleic acids are polymers ofsubunits or monomers, many of the modifications described below occur ata position which is repeated within a nucleic acid, e.g., a modificationof a base, or a phosphate moiety, or the non-linking O of a phosphatemoiety. In some cases the modification will occur at all of the subjectpositions in the nucleic acid but in many, and in fact in most, cases itwill not. By way of example, a modification may only occur at a 3′ or 5′terminal position, may only occur in a terminal region, e.g. at aposition on a terminal nucleotide or in the last 2, 3, 4, 5, or 10nucleotides of a strand. A modification may occur in a double strandregion, a single strand region, or in both. E.g., a phosphorothioatemodification at a non-linking O position may only occur at one or bothtermini, may only occur in a terminal regions, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand, or may occur in double strand and single strand regions,particularly at termini. Similarly, a modification may occur on thesense strand, antisense strand, or both. In some cases, the sense andantisense strand will have the same modifications or the same class ofmodifications, but in other cases the sense and antisense strand willhave different modifications, e.g., in some cases it may be desirable tomodify only one strand, e.g. the sense strand.

Two prime objectives for the introduction of modifications into iRNAagents is their stabilization towards degradation in biologicalenvironments and the improvement of pharmacological properties, e.g.pharmacodynamic properties, which are further discussed below. Othersuitable modifications to a sugar, base, or backbone of an iRNA agentare described in co-owned PCT Application No. PCT/US2004/01193, filedJan. 16, 2004. An iRNA agent can include a non-naturally occurring base,such as the bases described in co-owned PCT Application No.PCT/US2004/011822, filed Apr. 16, 2004. An iRNA agent can include anon-naturally occurring sugar, such as a non-carbohydrate cyclic carriermolecule. Exemplary features of non-naturally occurring sugars for usein iRNA agents are described in co-owned PCT Application No.PCT/US2004/11829 filed Apr. 16, 2003.

An iRNA agent can include an internucleotide linkage (e.g., the chiralphosphorothioate linkage) useful for increasing nuclease resistance. Inaddition, or in the alternative, an iRNA agent can include a ribosemimic for increased nuclease resistance. Exemplary internucleotidelinkages and ribose mimics for increased nuclease resistance aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can include ligand-conjugated monomer subunits andmonomers for oligonucleotide synthesis. Exemplary monomers are describedin co-owned U.S. application Ser. No. 10/916,185, filed on Aug. 10,2004.

An iRNA agent can have a ZXY structure, such as is described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

An iRNA agent can be complexed with an amphipathic moiety. Exemplaryamphipathic moieties for use with iRNA agents are described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

In another embodiment, the iRNA agent can be complexed to a deliveryagent that features a modular complex. The complex can include a carrieragent linked to one or more of (preferably two or more, more preferablyall three of): (a) a condensing agent (e.g., an agent capable ofattracting, e.g., binding, a nucleic acid, e.g., through ionic orelectrostatic interactions); (b) a fusogenic agent (e.g., an agentcapable of fusing and/or being transported through a cell membrane); and(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type. iRNA agents complexed to a delivery agent aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can have non-canonical pairings, such as between the senseand antisense sequences of the iRNA duplex. Exemplary features ofnon-canonical iRNA agents are described in co-owned PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004.

Enhanced Nuclease Resistance

An iRNA agent, e.g., an iRNA agent that targets RSV, can have enhancedresistance to nucleases.

For increased nuclease resistance and/or binding affinity to the target,an iRNA agent, e.g., the sense and/or antisense strands of the iRNAagent, can include, for example, 2′-modified ribose units and/orphosphorothioate linkages. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE andaminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino). It isnoteworthy that oligonucleotides containing only the methoxyethyl group(MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilitiescomparable to those modified with the robust phosphorothioatemodification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality.

Preferred substitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl,2′-C-allyl, and 2′-fluoro.

One way to increase resistance is to identify cleavage sites and modifysuch sites to inhibit cleavage, as described in co-owned U.S.Application No. 60/559,917, filed on May 4, 2004. For example, thedinucleotides 5′-UA-3′, 5′-UG-3′, 5′-CA-3′, 5′-UU-3′, or 5′-CC-3′ canserve as cleavage sites. Enhanced nuclease resistance can therefore beachieved by modifying the 5′ nucleotide, resulting, for example, in atleast one 5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide wherein theuridine is a 2′-modified nucleotide; at least one 5′-uridine-guanine-3′(5′-UG-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide; at least one 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide,wherein the 5′-cytidine is a 2′-modified nucleotide; at least one5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide; or at least one 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide. The iRNA agent can include at least 2, at least 3, at least4 or at least 5 of such dinucleotides. In certain embodiments, all thepyrimidines of an iRNA agent carry a 2′-modification, and the iRNA agenttherefore has enhanced resistance to endonucleases.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

The inclusion of furanose sugars in the oligonucleotide backbone canalso decrease endonucleolytic cleavage. An iRNA agent can be furthermodified by including a 3′ cationic group, or by inverting thenucleoside at the 3′-terminus with a 3′-3′ linkage. In anotheralternative, the 3′-terminus can be blocked with an aminoalkyl group,e.g., a 3′ C5-aminoalkyl dT. Other 3′ conjugates can inhibit 3′-5′exonucleolytic cleavage. While not being bound by theory, a 3′conjugate, such as naproxen or ibuprofen, may inhibit exonucleolyticcleavage by sterically blocking the exonuclease from binding to the3′-end of oligonucleotide. Even small alkyl chains, aryl groups, orheterocyclic conjugates or modified sugars (D-ribose, deoxyribose,glucose etc.) can block 3′-5′-exonucleases.

Similarly, 5′ conjugates can inhibit 5′-3′ exonucleolytic cleavage.While not being bound by theory, a 5′ conjugate, such as naproxen oribuprofen, may inhibit exonucleolytic cleavage by sterically blockingthe exonuclease from binding to the 5′-end of oligonucleotide. Evensmall alkyl chains, aryl groups, or heterocyclic conjugates or modifiedsugars (D-ribose, deoxyribose, glucose etc.) can block3′-5′-exonucleases.

An iRNA agent can have increased resistance to nucleases when a duplexediRNA agent includes a single-stranded nucleotide overhang on at leastone end. In preferred embodiments, the nucleotide overhang includes 1 to4, preferably 2 to 3, unpaired nucleotides. In a preferred embodiment,the unpaired nucleotide of the single-stranded overhang that is directlyadjacent to the terminal nucleotide pair contains a purine base, and theterminal nucleotide pair is a G-C pair, or at least two of the last fourcomplementary nucleotide pairs are G-C pairs. In further embodiments,the nucleotide overhang may have 1 or 2 unpaired nucleotides, and in anexemplary embodiment the nucleotide overhang is 5′-GC-3′. In preferredembodiments, the nucleotide overhang is on the 3′-end of the antisensestrand. In one embodiment, the iRNA agent includes the motif 5′-CGC-3′on the 3′-end of the antisense strand, such that a 2-nt overhang5′-GC-3′ is formed.

Thus, an iRNA agent can include modifications so as to inhibitdegradation, e.g., by nucleases, e.g., endonucleases or exonucleases,found in the body of a subject. These monomers are referred to herein asNRMs, or Nuclease Resistance promoting Monomers, the correspondingmodifications as NRM modifications. In many cases these modificationswill modulate other properties of the iRNA agent as well, e.g., theability to interact with a protein, e.g., a transport protein, e.g.,serum albumin, or a member of the RISC, or the ability of the first andsecond sequences to form a duplex with one another or to form a duplexwith another sequence, e.g., a target molecule.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent.

NRM modifications include some which can be placed only at the terminusand others which can go at any position. Some NRM modifications that caninhibit hybridization are preferably used only in terminal regions, andmore preferably not at the cleavage site or in the cleavage region of asequence which targets a subject sequence or gene, particularly on theantisense strand. They can be used anywhere in a sense strand, providedthat sufficient hybridization between the two strands of the ds iRNAagent is maintained. In some embodiments it is desirable to put the NRMat the cleavage site or in the cleavage region of a sense strand, as itcan minimize off-target silencing.

In most cases, the NRM modifications will be distributed differentlydepending on whether they are comprised on a sense or antisense strand.If on an antisense strand, modifications which interfere with or inhibitendonuclease cleavage should not be inserted in the region which issubject to RISC mediated cleavage, e.g., the cleavage site or thecleavage region (As described in Elbashir et al., 2001, Genes and Dev.15: 188, hereby incorporated by reference). Cleavage of the targetoccurs about in the middle of a 20 or 21 nt antisense strand, or about10 or 11 nucleotides upstream of the first nucleotide on the target mRNAwhich is complementary to the antisense strand. As used herein cleavagesite refers to the nucleotides on either side of the site of cleavage,on the target mRNA or on the iRNA agent strand which hybridizes to it.Cleavage region means the nucleotides within 1, 2, or 3 nucleotides ofthe cleavage site, in either direction.

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus,of a sequence which targets or a sequence which does not target asequence in the subject.

Tethered Ligands

The properties of an iRNA agent, including its pharmacologicalproperties, can be influenced and tailored, for example, by theintroduction of ligands, e.g. tethered ligands.

A wide variety of entities, e.g., ligands, can be tethered to an iRNAagent, e.g., to the carrier of a ligand-conjugated monomer subunit.Examples are described below in the context of a ligand-conjugatedmonomer subunit but that is only preferred, entities can be coupled atother points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe carrier. In preferred embodiments, the ligand is attached to thecarrier via an intervening tether. The ligand or tethered ligand may bepresent on the ligand-conjugated monomer when the ligand-conjugatedmonomer is incorporated into the growing strand. In some embodiments,the ligand may be incorporated into a “precursor” ligand-conjugatedmonomer subunit after a “precursor” ligand-conjugated monomer subunithas been incorporated into the growing strand. For example, a monomerhaving, e.g., an amino-terminated tether, e.g., TAP-(CH₂)_(n)NH₂ may beincorporated into a growing sense or antisense strand. In a subsequentoperation, i.e., after incorporation of the precursor monomer subunitinto the strand, a ligand having an electrophilic group, e.g., apentafluorophenyl ester or aldehyde group, can subsequently be attachedto the precursor ligand-conjugated monomer by coupling the electrophilicgroup of the ligand with the terminal nucleophilic group of theprecursor ligand-conjugated monomer subunit tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; nuclease-resistanceconferring moieties; and natural or unusual nucleobases. Generalexamples include lipophilic moleculeses, lipids, lectins, steroids(e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins, carbohydrates (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid), proteins, protein bindingagents, integrin targeting molecules, polycationics, peptides,polyamines, and peptide mimics.

The ligand may be a naturally occurring or recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.Examples of polyamino acids include polyamino acid is a polylysine(PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acidanhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinylether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamidecopolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers,or polyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic moieties, e.g., cationic lipid,cationic porphyrin, quaternary salt of a polyamine, or an alpha helicalpeptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a thyrotropin, melanotropin, surfactant proteinA, Mucin carbohydrate, a glycosylated polyaminoacid, transferrin,bisphosphonate, polyglutamate, polyaspartate, or an RGD peptide or RGDpeptide mimetic.

Ligands can be proteins, e.g., glycoproteins, lipoproteins, e.g. lowdensity lipoprotein (LDL), or albumins, e.g. human serum albumin (HSA),or peptides, e.g., molecules having a specific affinity for a co-ligand,or antibodies e.g., an antibody, that binds to a specified cell typesuch as a cancer cell, endothelial cell, or bone cell. Ligands may alsoinclude hormones and hormone receptors. They can also includenon-peptidic species, such as cofactors, multivalent lactose,multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine,multivalent mannose, or multivalent fucose. The ligand can be, forexample, a lipopolysaccharide, an activator of p38 MAP kinase, or anactivator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). Other molecules that can bind HSA can also beused as ligands. For example, neproxin or aspirin can be used. A lipidor lipid-based ligand can (a) increase resistance to degradation of theconjugate, (b) increase targeting or transport into a target cell orcell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another aspect, the ligand is a moiety, e.g., a vitamin or nutrient,which is taken up by a target cell, e.g., a proliferating cell. Theseare particularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include the B vitamins, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells.

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as that or antennapedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

5′-Phosphate Modifications

In preferred embodiments, iRNA agents are 5′ phosphorylated or include aphosphoryl analog at the 5′ prime terminus. 5′-phosphate modificationsof the antisense strand include those which are compatible with RISCmediated gene silencing. Suitable modifications include:5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure. Other suitable 5′-phosphate modifications will be known tothe skilled person.

The sense strand can be modified in order to inactivate the sense strandand prevent formation of an active RISC, thereby potentially reducingoff-target effects. This can be accomplished by a modification whichprevents 5′-phosphorylation of the sense strand, e.g., by modificationwith a 5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATPrequirements and small interfering RNA structure in the RNA interferencepathway. Cell 107, 309-321.) Other modifications which preventphosphorylation can also be used, e.g., simply substituting the 5′-OH byH rather than O-Me. Alternatively, a large bulky group may be added tothe 5′-phosphate turning it into a phosphodiester linkage.

Delivery of iRNA Agents to Tissues and Cells

Formulation

The iRNA agents described herein can be formulated for administration toa subject, preferably for administration locally to the lungs and nasalpassage (respiratory tissues) via inhalation or intranasallyadministration, or parenterally, e.g. via injection.

For ease of exposition, the formulations, compositions, and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions, and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.

A formulated iRNA agent composition can assume a variety of states. Insome examples, the composition is at least partially crystalline,uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20,or 10% water). In another example, the iRNA agent is in an aqueousphase, e.g., in a solution that includes water, this form being thepreferred form for administration via inhalation.

The aqueous phase or the crystalline compositions can be incorporatedinto a delivery vehicle, e.g., a liposome (particularly for the aqueousphase), or a particle (e.g., a microparticle as can be appropriate for acrystalline composition). Generally, the iRNA agent composition isformulated in a manner that is compatible with the intended method ofadministration.

An iRNA agent preparation can be formulated in combination with anotheragent, e.g., another therapeutic agent or an agent that stabilizes aniRNA agent, e.g., a protein that complexes with the iRNA agent to forman iRNP. Still other agents include chelators, e.g., EDTA (e.g., toremove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., abroad specificity RNAse inhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA agent preparation includes another iRNAagent, e.g., a second iRNA agent that can mediate RNAi with respect to asecond gene. Still other preparations can include at least three, five,ten, twenty, fifty, or a hundred or more different iRNA species. In someembodiments, the agents are directed to the same virus but differenttarget sequences. In another embodiment, each iRNA agents is directed toa different virus. As demonstrated in the Example, more than one viruscan be inhibited by co-administering two iRNA agents simultaneously, orat closely time intervals, each one directed to one of the viruses beingtreated.

Treatment Methods and Routes of Delivery

A composition that includes an iRNA agent of the present invention,e.g., an iRNA agent that targets RSV, can be delivered to a subject by avariety of routes. Exemplary routes include inhalation, intravenous,nasal, or oral delivery. The preferred means of administering the iRNAagents of the present invention is through direct administration to thelungs and nasal passage or systemically through parental administration.

An iRNA agent can be incorporated into pharmaceutical compositionssuitable for administration. For example, compositions can include oneor more iRNA agents and a pharmaceutically acceptable carrier. As usedherein the language “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including intranasal or intrapulmonary),oral or parenteral. Parenteral administration includes intravenous drip,subcutaneous, intraperitoneal or intramuscular injection.

In general, the delivery of the iRNA agents of the present invention isdone to achieve delivery into the subject to the site of infection. Thepreferred means of achieving this is through either a localadministration to the lungs or nasal passage, e.g. into the respiratorytissues via inhalation, nebulization or intranasal administration, orvia systemic administration, e.g. parental administration.

Formulations for inhalation or parenteral administration are well knownin the art. Such formulation may include sterile aqueous solutions whichmay also contain buffers, diluents and other suitable additives, anexample being PBS or Dextrose 5% in water. For intravenous use, thetotal concentration of solutes should be controlled to render thepreparation isotonic.

The active compounds disclosed herein are preferably administered to thelung(s) or nasal passage of a subject by any suitable means. Activecompounds may be administered by administering an aerosol suspension ofrespirable particles comprised of the active compound or activecompounds, which the subject inhales. The active compound can beaerosolized in a variety of forms, such as, but not limited to, drypowder inhalants, metered dose inhalants, or liquid/liquid suspensions.The respirable particles may be liquid or solid. The particles mayoptionally contain other therapeutic ingredients such as amiloride,benzamil or phenamil, with the selected compound included in an amounteffective to inhibit the reabsorption of water from airway mucoussecretions, as described in U.S. Pat. No. 4,501,729.

The particulate pharmaceutical composition may optionally be combinedwith a carrier to aid in dispersion or transport. A suitable carriersuch as a sugar (i.e., dextrose, lactose, sucrose, trehalose, mannitol)may be blended with the active compound or compounds in any suitableratio (e.g., a 1 to 1 ratio by weight).

Particles comprised of the active compound for practicing the presentinvention should include particles of respirable size, that is,particles of a size sufficiently small to pass through the mouth or noseand larynx upon inhalation and into the bronchi and alveoli of thelungs. In general, particles ranging from about 1 to 10 microns in size(more particularly, less than about 5 microns in size) are respirable.Particles of non-respirable size which are included in the aerosol tendto deposit in the throat and be swallowed, and the quantity ofnon-respirable particles in the aerosol is preferably minimized. Fornasal administration, a particle size in the range of 10-500 microns ispreferred to ensure retention in the nasal cavity.

Liquid pharmaceutical compositions of active compound for producing anaerosol may be prepared by combining the active compound with a suitablevehicle, such as sterile pyrogen free water. The hypertonic salinesolutions used to carry out the present invention are preferablysterile, pyrogen-free solutions, comprising from one to fifteen percent(by weight) of the physiologically acceptable salt, and more preferablyfrom three to seven percent by weight of the physiologically acceptablesalt.

Aerosols of liquid particles comprising the active compound may beproduced by any suitable means, such as with a pressure-driven jetnebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No.4,501,729. Nebulizers are commercially available devices which transformsolutions or suspensions of the active ingredient into a therapeuticaerosol mist either by means of acceleration of compressed gas,typically air or oxygen, through a narrow venturi orifice or by means ofultrasonic agitation.

Suitable formulations for use in nebulizers consist of the activeingredient in a liquid carrier, the active ingredient comprising up to40% w/w of the formulation, but preferably less than 20% w/w. Thecarrier is typically water (and most preferably sterile, pyrogen-freewater) or a dilute aqueous alcoholic solution, preferably made isotonic,but may be hypertonic with body fluids by the addition of, for example,sodium chloride. Optional additives include preservatives if theformulation is not made sterile, for example, methyl hydroxybenzoate,antioxidants, flavoring agents, volatile oils, buffering agents andsurfactants.

Aerosols of solid particles comprising the active compound may likewisebe produced with any solid particulate therapeutic aerosol generator.Aerosol generators for administering solid particulate therapeutics to asubject produce particles which are respirable and generate a volume ofaerosol containing a predetermined metered dose of a therapeutic at arate suitable for human administration. One illustrative type of solidparticulate aerosol generator is an insufflator. Suitable formulationsfor administration by insufflation include finely comminuted powderswhich may be delivered by means of an insufflator or taken into thenasal cavity in the manner of a snuff. In the insulator, the powder(e.g., a metered dose thereof effective to carry out the treatmentsdescribed herein) is contained in capsules or cartridges, typically madeof gelatin or plastic, which are either pierced or opened in situ andthe powder delivered by air drawn through the device upon inhalation orby means of a manually-operated pump. The powder employed in theinsufflator consists either solely of the active ingredient or of apowder blend comprising the active ingredient, a suitable powderdiluent, such as lactose, and an optional surfactant. The activeingredient typically comprises from 0.1 to 100 w/w of the formulation.

A second type of illustrative aerosol generator comprises a metered doseinhaler. Metered dose inhalers are pressurized aerosol dispensers,typically containing a suspension or solution formulation of the activeingredient in a liquefied propellant. During use these devices dischargethe formulation through a valve adapted to deliver a metered volume,typically from 10 to 200 ul, to produce a fine particle spray containingthe active ingredient. Suitable propellants include certainchlorofluorocarbon compounds, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.The formulation may additionally contain one or more co-solvents, forexample, ethanol, surfactants, such as oleic acid or sorbitan trioleate,antioxidant and suitable flavoring agents.

Administration can be provided by the subject or by another person,e.g., a caregiver. A caregiver can be any entity involved with providingcare to the human: for example, a hospital, hospice, doctor's office,outpatient clinic; a healthcare worker such as a doctor, nurse, or otherpractitioner; or a spouse or guardian, such as a parent. The medicationcan be provided in measured doses or in a dispenser which delivers ametered dose.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response. Inone embodiment, therapeutically effective amounts of two or more iRNAagents, each one directed to a different respiratory virus, e.g. RSV,are administered concurrently to a subject.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs.

The term “co-administration” refers to administering to a subject two ormore agents, and in particular two or more iRNA agents. The agents canbe contained in a single pharmaceutical composition and be administeredat the same time, or the agents can be contained in separate formulationand administered serially to a subject. So long as the two agents can bedetected in the subject at the same time, the two agents are said to beco-administered.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

Dosage. An iRNA agent can be administered at a unit dose less than about75 mg per kg of bodyweight, or less than about 70, 60, 50, 40, 30, 20,10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg ofbodyweight, and less than 200 nmol of iRNA agent (e.g., about 4.4×1016copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15,7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015nmol of iRNA agent per kg of bodyweight. The unit dose, for example, canbe administered by an inhaled dose or nebulization or by injection. Inone example, dosage ranges of 0.02-25 mg/kg is used.

Delivery of an iRNA agent directly to the lungs or nasal passage can beat a dosage on the order of about 1 mg to about 150 mg/nasal passage.

The dosage can be an amount effective to treat or prevent a disease ordisorder.

In one embodiment, the unit dose is administered once a day. In otherusage, a unit dose is administered twice the first day and then daily.Alternatively, unit dosing can be less than once a day, e.g., less thanevery 2, 4, 8 or 30 days. In another embodiment, the unit dose is notadministered with a frequency (e.g., not a regular frequency). Forexample, the unit dose may be administered a single time. Because iRNAagent mediated silencing can persist for several days afteradministering the iRNA agent composition, in many instances, it ispossible to administer the composition with a frequency of less thanonce per day, or, for some instances, only once for the entiretherapeutic regimen.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into an siRNA agent, or a DNA which encodes aniRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, orprecursor thereof). The maintenance dose or doses are generally lowerthan the initial dose, e.g., one-half less of the initial dose. Amaintenance regimen can include treating the subject with a dose ordoses ranging from 0.01 μg to 75 mg/kg of body weight per day, e.g., 70,60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or0.0005 mg per kg of bodyweight per day. The maintenance doses arepreferably administered no more than once every 5-14 days. Further, thetreatment regimen may last for a period of time which will varydepending upon the nature of the particular disease, its severity andthe overall condition of the patient. In preferred embodiments thedosage may be delivered no more than once per day, e.g., no more thanonce per 24, 36, 48, or more hours, e.g., no more than once every 5 or 8days. Following treatment, the patient can be monitored for changes inhis condition and for alleviation of the symptoms of the disease state.The dosage of the compound may either be increased in the event thepatient does not respond significantly to current dosage levels, or thedose may be decreased if an alleviation of the symptoms of the diseasestate is observed, if the disease state has been ablated, or ifundesired side-effects are observed.

In one embodiment, the iRNA agent pharmaceutical composition includes aplurality of iRNA agent species. The iRNA agent species can havesequences that are non-overlapping and non-adjacent with respect to anaturally occurring target sequence, e.g., a target sequence of the RSVgene. In another embodiment, the plurality of iRNA agent species isspecific for different naturally occurring target genes. For example, aniRNA agent that targets the P protein gene of RSV can be present in thesame pharmaceutical composition as an iRNA agent that targets adifferent gene, for example the N protein gene. In another embodiment,the iRNA agents are specific for different viruses, e.g. RSV.

The concentration of the iRNA agent composition is an amount sufficientto be effective in treating or preventing a disorder or to regulate aphysiological condition in humans. The concentration or amount of iRNAagent administered will depend on the parameters determined for theagent and the method of administration, e.g. nasal, buccal, orpulmonary. For example, nasal formulations tend to require much lowerconcentrations of some ingredients in order to avoid irritation orburning of the nasal passages. It is sometimes desirable to dilute anoral formulation up to 10-100 times in order to provide a suitable nasalformulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. It will also be appreciated thatthe effective dosage of an iRNA agent such as an siRNA agent used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays. For example, the subject can be monitoredafter administering an iRNA agent composition. Based on information fromthe monitoring, an additional amount of the iRNA agent composition canbe administered.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES

Designing Antiviral siRNAs Against RSV mRNA

siRNA against RSV P, N and L mRNA were synthesized chemically using knowprocedures. The siRNA sequences and some inhibition cross-subtypeactivity and IC50 values are listed (Table 1(a-c)).

In Vitro Assay and Virus Infection

Vero E6 cells were cultured to 80% confluency in DMEM containing 10%heat-inactivated FBS. For siRNA introduction, 4 μl of Transit-TKO wasadded to 50 μl of serum-free DMEM and incubated at room temperature for10 minutes. Then, indicated concentration of siRNA was added tomedia/TKO reagent respectively and incubated at room temperature for 10minutes. RNA mixture was added to 200 μl of DMEM containing 10% FBS andthen to cell monolayer. Cells were incubated at 37° C., 5% CO₂ for 6hours. RNA mixture was removed by gentle washing with 1× Hank's BalancedSalt Solutions (HBSS) and 300 plaque-forming units (pfu) per well ofRSV/A2 (MOI=30) was added to wells and adsorbed for 1 hour at 37° C., 5%CO₂. Virus was removed and cells were washed with 1×HBSS. Cells wereoverlaid with 1% methylcellulose in DMEM containing 10% FBS media, andincubated for 6 days at 37° C., 5% CO₂. Cells were immunostained forplaques using anti-F protein monoclonal antibody 131-2A.

siRNA Delivery and Virus Infection In Vivo

Pathogen-free 4 week old female BALB/c mice were purchased from Harlan.Mice were under anesthesia during infection and intranasal instillation(i.n.). Mice were immunized by intranasal instillation with indicatedamount of siRNA, either uncomplexed, or complexed with 5 ul Transit TKO.150 μg of Synagis (monoclonal antibody clone 143-6C, anti-RSV F protein)and Mouse Isotype control (IgG1) were administered intraperitoneal(i.p.) four hours prior to RSV challenge (10⁶ PFU of RSV/A2). Ten miceper group were used. Animal weights were monitored at days 0, 2, 4, and6 post-infection. Lungs were harvested at day 6 post-infection, andassayed for RSV by immunostaining plaque assay.

Immunostaining Plaque Assay

24-well plates of Vero E6 cells were cultured to 90% confluency in DMEMcontaining 10% heat inactivated FBS. Mice lungs were homogenized withhand-held homogenizer in 1 ml sterile Dulbecco's PBS (D-PBS) and 10 folddiluted in serum-free DMEM. Virus containing lung lysate dilutions wereplated onto 24 well plates in triplicate and adsorbed for 1 hour at 37°C., 5% CO₂. Wells were overlaid with 1% methylcellulose in DMEMcontaining 10% FBS. Then, plates were incubated for 6 days at 37° C., 5%CO₂. After 6 days, overlaid media was removed and cells were fixed inacetone:methanol (60:40) for 15 minutes. Cells were blocked with 5% dryMilk/PBS for 1 hour at 37° C. 1:500 dilution of anti-RSV F proteinantibody (131-2A) was added to wells and incubated for 2 hours at 37° C.Cells were washed twice in PBS/0.5% Tween 20. 1:500 dilution of goatanti-mouse IgG-Alkaline Phosphatase was added to wells and incubated for1 hour at 37° C. Cells were washed twice in PBS/0.5% Tween 20. Reactionwas developed using Vector's Alkaline Phosphatase substrate kit II(Vector Black), and counterstained with Hematoxylin. Plaques werevisualized and counted using an Olympus Inverted microscope.

Treatment Assay

Mice were challenged with RSV (10⁶ PFU of RSV/A2) by intranasalinstillation at day 0 and treated with 50 ug of indicated siRNA,delivered by intranasal instillation, at the indicated times (day 1-4post viral challenge). 3-5 mice per group were used and viral titerswere measured from lung lysates at day 5 post viral challenge, aspreviously described.

In Vitro Inhibition of RSV Using iRNA Agents.

iRNA agents provided in Table 1(a-c) were tested for anti-RSV activityin a plaque formation assay as described above (FIG. 1). Each column(bar) represents an iRNA agent provided in Table 1(a-c), e.g. column 1is the first agent in Table 1a, second column is the second agent and soon. Active iRNA agents were identified by the % of virus remaining.Several agents were identified that showed as much as 90% inhibition.The results are summarized in Table 1(a-c).

In vitro dose response inhibition of RSV using iRNA agents wasdetermined. Examples of active agents from Table 1 were tested foranti-RSV activity in a plaque formation assay as described above at fourconcentrations. A dose dependent response was found with active iRNAagent tested (FIG. 2) and is summarized in Tables 1(a-c).

In vitro inhibition of RSV B subtype using iRNA agents was tested asdescribed above. iRNA agents provided in FIG. 2 were tested for anti-RSVactivity against subtype B (FIG. 3). RSV subtype B was inhibited by theiRNA agents tested to varying degrees and is summarized in Table 1(a-c).

In Vivo Inhibition of RSV Using iRNA Agents.

In vivo inhibition of RSV using AL1729 and AL1730 was tested asdescribed above. Agents as described in FIG. 4 were tested for anti-RSVactivity in a mouse model. The iRNA agents were effective at reducingviral titers in vivo and more effective than a control antibody (Mab143-6c, a mouse IgG1 Ab that is approved for RSV treatment).

AL1730 was tested for dose dependent activity using the methods providedabove. The agents showed a dose dependent response (FIG. 5).

iRNA agents showing in vitro activity were tested for anti-RSV activityin vivo as outlined above. Several agents showed a reduction in viraltiters of >4 logs when given prophylactically (FIG. 6).

iRNA agents showing in vitro and/or in vivo activity were tested foranti-RSV activity in vivo as in the treatment protocol outlined above.Several agents showed a reduction in viral titers of 2-3 logs (FIG. 7)when given 1-2 days following viral infection.

Sequence Analysis of Isolates Across Target Sequence

Method: Growth of isolates and RNA isolation: Clinical isolates from RSVinfected patients were obtained from Larry Anderson at the CDC inAtlanta Ga. (4 strains) and John DeVincenzo at the University of Tenn.,Memphis (15 strains). When these were grown in HEp-2, human epithelialcells (ATCC, Cat# CCL-23) cells, it was noted that the 4 isolates fromGeorgia were slower growing than the 15 strains from Tennessee; hence,these were processed and analyzed separately. The procedure is brieflydescribed as follows:

Vero E6, monkey kidney epithelial cells (ATCC, Cat# CRL-1586) were grownto 95% confluency and infected with a 1/10 dilution of primary isolates.The virus was absorbed for 1 hour at 37° C., then cells weresupplemented with D-MEM and incubated at 37° C. On a daily basis, cellswere monitored for cytopathic effect (CPE) by light microscopy. At 90%CPE, the cells were harvested by scraping and pelleted by centrifugationat 3000 rpm for 10 minutes. RNA preparations were performed by standardprocedures according to manufacturer's protocol.

Amplification of RSV N gene: Viral RNAs were collected post-infectionand used as templates in PCR reactions, using primers that hybridizeupstream and downstream of the ALDP-2017 target site to amplify an ˜450bp fragment. Total RNA was denatured at

65° C. for 5 minutes in the presence of forward and reverse RSV N geneprimers, stored on ice, and then reverse-transcribed with SuperscriptIII (Invitrogen) for 60 minutes at 55° C. and for 15 minutes at 70° C.PCR products were analyzed by gel electrophoresis on a 1% agarose geland purified by standard protocols.

Results: Sequence analysis of the first 15 isolates confirmed that thetarget site for ALDP-2017 was completely conserved across every strain.Importantly, this conservation was maintained across the diversepopulations, which included isolates from both RSV A and B subtypes.Interestingly, when the 4 slower-growing isolates were analyzed, weobserved that one of the 4 (LAP6824) had a single base mutation in theALDP-2017 recognition site. This mutation changed the coding sequence atposition 13 of the RSV N gene in this isolate from an A to a G.

Conclusions: From 19 patient isolates, the sequence of the RSV N gene atthe target site for ALDP-2017 has been determined. In 18 of 19 cases(95%), the recognition element for ALDP-2017 is 100% conserved. In oneof the isolates, there is a single base alteration changing thenucleotide at position 13 from an A to a G within the RSV N gene. Thisalteration creates a single G:U wobble between the antisense strand ofALDP-2017 and the target sequence. Based on an understanding of thehybridization potential of such a G:U wobble, it is predicted thatALDP-2017 will be effective in silencing the RSV N gene in this isolate.

Silencing Data on Isolates

Methods: Vero E6 cells were cultured to 80% confluency in DMEMcontaining 10% heat-inactivated FBS. For siRNA introduction, 4 μl ofTransit-TKO was added to 50 μl of serum-free DMEM and incubated at roomtemperature for 10 minutes. Then, indicated concentration of siRNA wasadded to media/TKO reagent respectively and incubated at roomtemperature for 10 minutes. RNA mixture was added to 200 μl of DMEMcontaining 10% FBS and then to cell monolayer. Cells were incubated at37° C., 5% CO₂ for 6 hours. RNA mixture was removed by gentle washingwith 1× Hank's Balanced Salt Solutions (HBSS) and 300 plaque-formingunits (pfu) per well of RSV/A2 (MOI=30) was added to wells and adsorbedfor 1 hour at 37° C., 5% CO₂. Virus was removed and cells were washedwith 1×HBSS. Cells were overlaid with 1% methylcellulose in DMEMcontaining 10% FBS media, and incubated for 6 days at 37° C., 5% CO₂.Cells were immunostained for plaques using anti-F protein monoclonalantibody 131-2A.

Results: Silencing was seen for all isolates (Table 2)

TABLE 2 2017 2153 Isolate % plaques % plaques name remaining remainingRSV/A2 4.49 80.34 RSV/96 5.36 87.50 RSV/87 10.20 79.59 RSV/110 5.4181.08 RSV/37 4.80 89.60 RSV/67 2.22 91.67 RSV/121 6.25 82.50 RSV/31 4.0396.77 RSV/38 2.00 92.67 RSV/98 5.13 91.03 RSV/124 3.74 90.37 RSV/95 7.3264.02 RSV/32 5.45 92.73 RSV/91 8.42 95.79 RSV/110 12.07 94.83 RSV/541.90 89.87 RSV/53 7.41 94.07 RSV/33 7.69 95.19

Conclusion: All clinical isolates tested were specifically inhibited bysiRNA 2017 by greater than 85%. No isolates were significantly inhibitedthe mismatch control siRNA 2153.

Silencing in Plasmid Based Assay

Method: A 24-well plate is seeded with HeLa S6 cells and grown to 80%confluence. For each well, mix 1 ug of RSV N-V5 plasmid with siRNA (atindicated concentration), in 50 ul OPTI-MEM and add to Lipofectamine2000 (Invitrogen)-Optimem mixture prepared according to manufacturer'sinstructions, and let sit 20 minutes at r.t. to form complex. Addcomplex to cells and incubate 37° C. overnight. Remove the media, washthe cells with PBS and lyse with 50 ul Lysis buffer (RIPA buffer (50 mMTris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% Na deoxycholate, 1% NP-40,0.05% SDS) for 1-2 min. Lysates are analysed using Inhibition isquantified by measuring the level of RSV protein in cell lysates,detected by western blotting with an anti-V5 antibody

Results: Transient plasmid expression was shown to be an effective assayfor RNAi agents (Table 3).

TABLE 3 Protein % Activity % 1 ALDP2017  10 nM 0 100 2  1 nM 0 100 3 100pM 0 100 4  10 pM 11.78 88.22 5  1 pM 70.63 29.37 6 100 fM 72.7 27.3 7Control PBS 100 0 8 2153  10 nM 94.54 4.5

Conclusions: siRNA 2017 specifically and dose dependently inhibits theproduction of RSV N protein when transiently cotranfected with plasmidexpressing the RSV N gene. Inhibition is not observed with mismatchcontrol siRNA 2153.

Silencing of RSV Via Aerosol Delivery of siRNA

Method: A 2 mg/ml solution of ALDP-1729 or ALDP-1730 is delivered vianebulization using an aerosol device for a total of 60 sec. Viral isprepared from lung as described above and measured by an ELISA insteadof a plaque assay. The ELISA measures the concentration of the RSV Nprotein in cells infected with virus obtained from mouse lung lysates.

ELISA

Lung lysate is diluted 1:1 with carbonate-bicarbonate buffer (NaHCO₃ pH9.6) to a working concentration of 6-10 μg/100 μL, added to each testwell and incubated at 37° C. for 1 hour or overnight at 4° C. Wellswashed 3× with PBS/0.5% Tween 20 then blocked with 5% dry milk/PBS for 1hour at 37° C. or overnight at 4° C. Primary antibody (F proteinpositive control=clone 131-2A; G protein positive control=130-2G;negative control=normal IgG1□, (BD Pharmingen, cat. #553454, test sera,or hybridoma supernatant) is added to wells at 1:1000 and incubated at37° C. for 1 hour or overnight at 4° C. Wells washed 3× with PBS/0.5%Tween 20. Secondary antibody (Goat Anti-mouse IgG (H+L) wholemolecule-alkaline phosphatase conjugated) diluted 1:1000 to wells (100μl/well) is added and incubated at 37° C. for 1 hour or overnight at 4°C. Wash 3× with PBS/0.5% Tween 20 then add Npp (Sigmafast) substrateSigma Aldrich N2770 accordingly to manufacturers instructions. Add 200μl of substrate/well and incubate for 10-15. Measure absorbance at OD405/495.

Conclusion: Delivery of RSV specific siRNA decreases the levels of RSV Nprotein in mouse lungs as compared to the mismatch control siRNA (FIG. 8a-b).

In Vivo Inhibition at Day-3-Prophylaxis

Method: In vivo prophylaxis was tested using the in vivo methoddescribed above except that the siRNA is delivered at different timesprior to infection with RSV from 3 days before to 4 hrs before.

Results: siRNA delivered intranasally up to 3 days prior to viralchallenge show significant silencing in vivo (FIG. 9).

Nebulization of AL-DP 2017 with Pari eFlow® Device

Droplet Size and Analytical Integrity

A 75 mg/ml solution of AL-DP 2017 (in 2 mls of PBS) was filled into thePari eFlow® electronic device and run until nebulization was completedand all aerosol was collected and allowed to condense in a polypropylenetube. Aliquots of material post nebulization were analyzed to determinegeometric droplet size distribution by laser diffraction (MalvernMasterSizerX) under standard conditions. Aliquots of material pre andpost nebulization were analyzed to determine analytical integrity by astability indicating anion exchange HPLC method.

Results: Aerosolized AL-DP 2017 had a Mass Median Diameter (MMD) of 3.1□m, a Geometric Standard Deviation (GSD) of 1.6 and a total respirablefraction of 85% (ie, % particles<5 □m) confirming that a 75 mg/mlsolution could be aerosolized to yield respirable material withappropriate particle size. Comparison to control samples of AL-DP 2017formulation which were not nebulized showed matching chromatograms,demonstrating that the oligonucleotide can be nebulized by eFlow®without degradation.

Biological Activity: A 25 mg/ml solution of AL-DP 2017 (in 1 mls of PBS)was prepared, 100 μl was removed (pre-nebulization aliquot) prior tonebulization with the Pari eFlow® electronic device, and 500 ul of thenebulized solution was collected after condensing by passage over an icebath into a chilled 50 ml conical tube (post-nebulization aliquot).Serial dilutions of both aliquots were tested in our in vitrotransfection/infection plaque assay as previously described with theexception that siRNA is complexed with lipofetamine-2000.

Results: siRNA pre and post nebulization efficiently inhibited RSV viralreplication in a Vero cell plaque assay. The degree of inhibition wasalmost identical between the two samples and showed a dose responseleading to >80% silencing at the highest siRNA concentrations confirmingthat nebulized AL-DP 2017 maintains biological activity (FIG. 10).

TABLE 1a RSV L gene % inh % inh % inh % inh % inh Actual WhiteheadRSV A2 RSV A2 RSV A2 RSV A2 RSVB start Start Pos Sense SEQ ID NOAntisense SEQ ID NO AL-DP # (5 nM) 500 pM 50 pM 5 pM (5-nM) 3 1GGAUCCCAUUAUUAAUGGAdTdT 1 UCCAUUAAUAAUGGGAUCCdTdT 2 AL-DP-2024 92 4 2GAUCCCAUUAUUAAUGGAAdTdT 3 UUCCAUUAAUAAUGGGAUCdTdT 4 AL-DP-2026 82 49 47AGUUAUUUAAAAGGUGUUAdTdT 5 UAACACCUUUUAAAUAACUdTdT 6 AL-DP-2116 50 48GUUAUUUAAAAGGUGUUAUdTdT 7 AUAACACCUUUUAAAUAACdTdT 8 AL-DP-2117 53 51AUUUAAAAGGUGUUAUCUCdTdT 9 GAGAUAACACCUUUUAAAUdTdT 10 AL-DP-2118 55 53UUAAAAGGUGUUAUCUCUUdTdT 11 AAGAGAUAACACCUUUUAAdTdT 12 AL-DP-2119 156 154AAGUCCACUACUAGAGCAUdTdT 13 AUGCUCUAGUAGUGGACUUdTdT 14 AL-DP-2027 86 157155 AGUCCACUACUAGAGCAUAdTdT 15 UAUGCUCUAGUAGUGGACUdTdT 16 AL-DP-2028 90158 156 GUCCACUACUAGAGCAUAUdTdT 17 AUAUGCUCUAGUAGUGGACdTdT 18 AL-DP-202989 159 157 UCCACUACUAGAGCAUAUGdTdT 19 CAUAUGCUCUAGUAGUGGAdTdT 20AL-DP-2030 86 341 339 GAAGAGCUAUAGAAAUAAGdTdT 21 CUUAUUUCUAUAGCUCUUCdTdT22 AL-DP-2120 344 342 GAGCUAUAGAAAUAAGUGAdTdT 23 UCACUUAUUUCUAUAGCUCdTdT24 AL-DP-2121 347 345 CUAUAGAAAUAAGUGAUGUdTdT 25 ACAUCACUUAUUUCUAUAGdTdT26 AL-DP-2031 15 554 552 UCAAAACAACACUCUUGAAdTdT 27UUCAAGAGUGUUGUUUUGAdTdT 28 AL-DP-2122 1004 1002 UAGAGGGAUUUAUUAUGUCdTdT29 GACAUAAUAAAUCCCUCUAdTdT 30 AL-DP-2123 1408 1406AUAAAAGGGUUUGUAAAUAdTdT 31 UAUUUACAAACCCUUUUAUdTdT 32 AL-DP-2124 18671865 CUCAGUGUAGGUAGAAUGUdTdT 33 ACAUUCUACCUACACUGAGdTdT 34 AL-DP-2032 901868 1866 UCAGUGUAGGUAGAAUGUUdTdT 35 AACAUUCUACCUACACUGAdTdT 36AL-DP-2033 84 1869 1867 CAGUGUAGGUAGAAUGUUUdTdT 37AAACAUUCUACCUACACUGdTdT 38 AL-DP-2034 86 1870 1868AGUGUAGGUAGAAUGUUUGdTdT 39 CAAACAUUCUACCUACACUdTdT 40 AL-DP-2112 18711869 GUGUAGGUAGAAUGUUUGCdTdT 41 GCAAACAUUCUACCUACACdTdT 42 AL-DP-21131978 1976 ACAAGAUAUGGUGAUCUAGdTdT 43 CUAGAUCACCAUAUCUUGUdTdT 44AL-DP-2035 89 2104 2102 AGCAAAUUCAAUCAAGCAUdTdT 45AUGCUUGAUUGAAUUUGCUdTdT 46 AL-DP-2036 87 2105 2103GCAAAUUCAAUCAAGCAUUdTdT 47 AAUGCUUGAUUGAAUUUGCdTdT 48 AL-DP-2037 91 22902288 GAUGAACAAAGUGGAUUAUdTdT 49 AUAAUCCACUUUGUUCAUCdTdT 50 AL-DP-2038 112384 2382 UAAUAUCUCUCAAAGGGAAdTdT 51 UUCCCUUUGAGAGAUAUUAdTdT 52AL-DP-2125 2386 2384 AUAUCUCUCAAAGGGAAAUdTdT 53 AUUUCCCUUUGAGAGAUAUdTdT54 AL-DP-2126 2387 2385 UAUCUCUCAAAGGGAAAUUdTdT 55AAUUUCCCUUUGAGAGAUAdTdT 56 AL-DP-2127 2485 2483 CAUGCUCAAGCAGAUUAUUdTdT57 AAUAAUCUGCUUGAGCAUGdTdT 58 AL-DP-2039 87 2487 2485UGCUCAAGCAGAUUAUUUGdTdT 59 CAAAUAAUCUGCUUGAGCAdTdT 60 AL-DP-2040 88 25072505 UAGCAUUAAAUAGCCUUAAdTdT 61 UUAAGGCUAUUUAAUGCUAdTdT 62 AL-DP-2041 9676 73 69 94 2508 2506 AGCAUUAAAUAGCCUUAAAdTdT 63 UUUAAGGCUAUUUAAUGCUdTdT64 AL-DP-2114 2509 2507 GCAUUAAAUAGCCUUAAAUdTdT 65AUUUAAGGCUAUUUAAUGCdTdT 66 AL-DP-2042 96 98 97 97 90 2510 2508CAUUAAAUAGCCUUAAAUUdTdT 67 AAUUUAAGGCUAUUUAAUGdTdT 68 AL-DP-2043 97 8679 75 94 2765 2763 UAUUAUGCAGUUUAAUAUUdTdT 69 AAUAUUAAACUGCAUAAUAdTdT 70AL-DP-2044 97 79 72 67 84 2767 2765 UUAUGCAGUUUAAUAUUUAdTdT 71UAAAUAUUAAACUGCAUAAdTdT 72 AL-DP-2045 15 3283 3281AAAAGUGCACAACAUUAUAdTdT 73 UAUAAUGUUGUGCACUUUUdTdT 74 AL-DP-2128 32843282 AAAGUGCACAACAUUAUACdTdT 75 GUAUAAUGUUGUGCACUUUdTdT 76 AL-DP-2046 9494 91 91 93 3338 3336 AUAUAGAACCUACAUAUCCdTdT 77 GGAUAUGUAGGUUCUAUAUdTdT78 AL-DP-2047 87 3339 3337 UAUAGAACCUACAUAUCCUdTdT 79AGGAUAUGUAGGUUCUAUAdTdT 80 AL-DP-2048 84 3365 3363UAAGAGUUGUUUAUGAAAGdTdT 81 CUUUCAUAAACAACUCUUAdTdT 82 AL-DP-2129 40214019 ACAGUCAGUAGUAGACCAUdTdT 83 AUGGUCUACUACUGACUGUdTdT 84 AL-DP-2049 244022 4020 CAGUCAGUAGUAGACCAUGdTdT 85 CAUGGUCUACUACUGACUGdTdT 86AL-DP-2050 15 4023 4021 AGUCAGUAGUAGACCAUGUdTdT 87ACAUGGUCUACUACUGACUdTdT 88 AL-DP-2051 87 4024 4022GUCAGUAGUAGACCAUGUGdTdT 89 CACAUGGUCUACUACUGACdTdT 90 AL-DP-2052 96 8476 69 87 4025 4023 UCAGUAGUAGACCAUGUGAdTdT 91 UCACAUGGUCUACUACUGAdTdT 92AL-DP-2053 92 84 79 76 74 4037 4035 CAUGUGAAUUCCCUGCAUCdTdT 93GAUGCAGGGAAUUCACAUGdTdT 94 AL-DP-2054 97 79 78 69 96 4038 4036AUGUGAAUUCCCUGCAUCAdTdT 95 UGAUGCAGGGAAUUCACAUdTdT 96 AL-DP-2055 88 40394037 UGUGAAUUCCCUGCAUCAAdTdT 97 UUGAUGCAGGGAAUUCACAdTdT 98 AL-DP-2056 164040 4038 GUGAAUUCCCUGCAUCAAUdTdT 99 AUUGAUGCAGGGAAUUCACdTdT 100AL-DP-2115 4043 4041 AAUUCCCUGCAUCAAUACCdTdT 101 GGUAUUGAUGCAGGGAAUUdTdT102 AL-DP-2057 94 91 86 79 69 4051 4049 GCAUCAAUACCAGCUUAUAdTdT 103UAUAAGCUGGUAUUGAUGCdTdT 104 AL-DP-2058 86 4052 4050CAUCAAUACCAGCUUAUAGdTdT 105 CUAUAAGCUGGUAUUGAUGdTdT 106 AL-DP-2059 914057 4055 AUACCAGCUUAUAGAACAAdTdT 107 UUGUUCUAUAAGCUGGUAUdTdT 108AL-DP-2060 92 4058 4056 UACCAGCUUAUAGAACAACdTdT 109GUUGUUCUAUAAGCUGGUAdTdT 110 AL-DP-2061 88 4059 4057ACCAGCUUAUAGAACAACAdTdT 111 UGUUGUUCUAUAAGCUGGUdTdT 112 AL-DP-2062 95 7978 72 94 4060 4058 CCAGCUUAUAGAACAACAAdTdT 113 UUGUUGUUCUAUAAGCUGGdTdT114 AL-DP-2063 90 4061 4059 CAGCUUAUAGAACAACAAAdTdT 115UUUGUUGUUCUAUAAGCUGdTdT 116 AL-DP-2064 94 86 76 67 83 4067 4065AUAGAACAACAAAUUAUCAdTdT 117 UGAUAAUUUGUUGUUCUAUdTdT 118 AL-DP-2065 914112 4110 UAUUAACAGAAAAGUAUGGdTdT 119 CCAUACUUUUCUGUUAAUAdTdT 120AL-DP-2130 4251 4249 UGAGAUACAUUUGAUGAAAdTdT 121 UUUCAUCAAAUGUAUCUCAdTdT122 AL-DP-2066 86 4252 4250 GAGAUACAUUUGAUGAAACdTdT 123GUUUCAUCAAAUGUAUCUCdTdT 124 AL-DP-2067 92 4254 4252GAUACAUUUGAUGAAACCUdTdT 125 AGGUUUCAUCAAAUGUAUCdTdT 126 AL-DP-2068 934255 4253 AUACAUUUGAUGAAACCUCdTdT 127 GAGGUUUCAUCAAAUGUAUdTdT 128AL-DP-2069 89 4256 4254 UACAUUUGAUGAAACCUCCdTdT 129GGAGGUUUCAUCAAAUGUAdTdT 130 AL-DP-2074 4313 4311 AAGUGAUACAAAAACAGCAdTdT131 UGCUGUUUUUGUAUCACUUdTdT 132 AL-DP-2131 4314 4312AGUGAUACAAAAACAGCAUdTdT 133 AUGCUGUUUUUGUAUCACUdTdT 134 AL-DP-2132 43164314 UGAUACAAAAACAGCAUAUdTdT 135 AUAUGCUGUUUUUGUAUCAdTdT 136 AL-DP-21334473 4471 UUUAAGUACUAAUUUAGCUdTdT 137 AGCUAAAUUAGUACUUAAAdTdT 138AL-DP-2075 4474 4472 UUAAGUACUAAUUUAGCUGdTdT 139 CAGCUAAAUUAGUACUUAAdTdT140 AL-DP-2076 4475 4473 UAAGUACUAAUUUAGCUGGdTdT 141CCAGCUAAAUUAGUACUUAdTdT 142 AL-DP-2077 4476 4474 AAGUACUAAUUUAGCUGGAdTdT143 UCCAGCUAAAUUAGUACUUdTdT 144 AL-DP-2078 4477 4475AGUACUAAUUUAGCUGGACdTdT 145 GUCCAGCUAAAUUAGUACUdTdT 146 AL-DP-2079 44784476 GUACUAAUUUAGCUGGACAdTdT 147 UGUCCAGCUAAAUUAGUACdTdT 148 AL-DP-20804480 4478 ACUAAUUUAGCUGGACAUUdTdT 149 AAUGUCCAGCUAAAUUAGUdTdT 150AL-DP-2081 4483 4481 AAUUUAGCUGGACAUUGGAdTdT 151 UCCAAUGUCCAGCUAAAUUdTdT152 AL-DP-2082 4484 4482 AUUUAGCUGGACAUUGGAUdTdT 153AUCCAAUGUCCAGCUAAAUdTdT 154 AL-DP-2083 4486 4484 UUAGCUGGACAUUGGAUUCdTdT155 GAAUCCAAUGUCCAGCUAAdTdT 156 AL-DP-2084 4539 4537UUUUGAAAAAGAUUGGGGAdTdT 157 UCCCCAAUCUUUUUCAAAAdTdT 158 AL-DP-2134 45404538 UUUGAAAAAGAUUGGGGAGdTdT 159 CUCCCCAAUCUUUUUCAAAdTdT 160 AL-DP-21354542 4540 UGAAAAAGAUUGGGGAGAGdTdT 161 CUCUCCCCAAUCUUUUUCAdTdT 162AL-DP-2136 4543 4541 GAAAAAGAUUGGGGAGAGGdTdT 163 CCUCUCCCCAAUCUUUUUCdTdT164 AL-DP-2137 4671 4669 UAUGAACACUUCAGAUCUUdTdT 165AAGAUCUGAAGUGUUCAUAdTdT 166 AL-DP-2085 4672 4670 AUGAACACUUCAGAUCUUCdTdT167 GAAGAUCUGAAGUGUUCAUdTdT 168 AL-DP-2086 4867 4865UGCCCUUGGGUUGUUAACAdTdT 169 UGUUAACAACCCAAGGGCAdTdT 170 AL-DP-2087 48684866 GCCCUUGGGUUGUUAACAUdTdT 171 AUGUUAACAACCCAAGGGCdTdT 172 AL-DP-20885544 5542 UAUAGCAUUCAUAGGUGAAdTdT 173 UUCACCUAUGAAUGCUAUAdTdT 174AL-DP-2089 5545 5543 AUAGCAUUCAUAGGUGAAGdTdT 175 CUUCACCUAUGAAUGCUAUdTdT176 AL-DP-2090 5546 5544 UAGCAUUCAUAGGUGAAGGdTdT 177CCUUCACCUAUGAAUGCUAdTdT 178 AL-DP-2091 5550 5548 AUUCAUAGGUGAAGGAGCAdTdT179 UGCUCCUUCACCUAUGAAUdTdT 180 AL-DP-2092 5640 5638UUGCAAUGAUCAUAGUUUAdTdT 181 UAAACUAUGAUCAUUGCAAdTdT 182 AL-DP-2093 56415639 UGCAAUGAUCAUAGUUUACdTdT 183 GUAAACUAUGAUCAUUGCAdTdT 184 AL-DP-20945642 5640 GCAAUGAUCAUAGUUUACCdTdT 185 GGUAAACUAUGAUCAUUGCdTdT 186AL-DP-2095 5643 5641 CAAUGAUCAUAGUUUACCUdTdT 187 AGGUAAACUAUGAUCAUUGdTdT188 AL-DP-2096 5644 5642 AAUGAUCAUAGUUUACCUAdTdT 189UAGGUAAACUAUGAUCAUUdTdT 190 AL-DP-2097 5645 5643 AUGAUCAUAGUUUACCUAUdTdT191 AUAGGUAAACUAUGAUCAUdTdT 192 AL-DP-2098 5647 5645GAUCAUAGUUUACCUAUUGdTdT 193 CAAUAGGUAAACUAUGAUCdTdT 194 AL-DP-2138 56485646 AUCAUAGUUUACCUAUUGAdTdT 195 UCAAUAGGUAAACUAUGAUdTdT 196 AL-DP-21395649 5647 UCAUAGUUUACCUAUUGAGdTdT 197 CUCAAUAGGUAAACUAUGAdTdT 198AL-DP-2140 5650 5648 CAUAGUUUACCUAUUGAGUdTdT 199 ACUCAAUAGGUAAACUAUGdTdT200 AL-DP-2099 5651 5649 AUAGUUUACCUAUUGAGUUdTdT 201AACUCAAUAGGUAAACUAUdTdT 202 AL-DP-2100 5752 5750 CAUUGGUCUUAUUUACAUAdTdT203 UAUGUAAAUAAGACCAAUGdTdT 204 AL-DP-2101 5754 5752UUGGUCUUAUUUACAUAUAdTdT 205 UAUAUGUAAAUAAGACCAAdTdT 206 AL-DP-2102 57555753 UGGUCUUAUUUACAUAUAAdTdT 207 UUAUAUGUAAAUAAGACCAdTdT 208 AL-DP-21035756 5754 GGUCUUAUUUACAUAUAAAdTdT 209 UUUAUAUGUAAAUAAGACCdTdT 210AL-DP-2141 5919 5917 AUAUCAUGCUCAAGAUGAUdTdT 211 AUCAUCUUGAGCAUGAUAUdTdT212 AL-DP-2142 5920 5918 UAUCAUGCUCAAGAUGAUAdTdT 213UAUCAUCUUGAGCAUGAUAdTdT 214 AL-DP-2104 5934 5932 UGAUAUUGAUUUCAAAUUAdTdT215 UAAUUUGAAAUCAAUAUCAdTdT 216 AL-DP-2105 6016 6014UACUUAGUCCUUACAAUAGdTdT 217 CUAUUGUAAGGACUAAGUAdTdT 218 AL-DP-2106 60196017 UUAGUCCUUACAAUAGGUCdTdT 219 GACCUAUUGUAAGGACUAAdTdT 220 AL-DP-21076020 6018 UAGUCCUUACAAUAGGUCCdTdT 221 GGACCUAUUGUAAGGACUAdTdT 222AL-DP-2108 6252 6250 AUAUUCUAUAGCUGGACGUdTdT 223 ACGUCCAGCUAUAGAAUAUdTdT224 AL-DP-2109 6253 6251 UAUUCUAUAGCUGGACGUAdTdT 225UACGUCCAGCUAUAGAAUAdTdT 226 AL-DP-2110 6254 6252 AUUCUAUAGCUGGACGUAAdTdT227 UUACGUCCAGCUAUAGAAUdTdT 228 AL-DP-2111

TABLE 1b RSV P gene % % % % % inhibi- inhibi- inhibi- inhibi- inhibi-tion tion tion tion Actual Start SEQ ID SEQ ID tion RSV A2 RSV A2 RSV A2RSV B start Pos Sense  NO Antisense  NO AL-DP # (5 nM) 500 pM 50 pM 5 pM(5 nM) 55 53 AAAUUCCUAGAA 229  UUAUUGAUUCUA 230 AL-DP- 3 UCAAUAAdTdTGGAAUUUdTdT 232 2000 56 54 AAUUCCUAGAAU 231 UUUAUUGAUUCUA 234 AL-DP- 4CAAUAAAdTdT GGAAUUdTdT 2001 58 56 UUCCUAGAAUCA 233 CCUUUAUUGAUUC 236AL-DP- 7 AUAAAGGdTdT UAGGAAdTdT 2002 59 57 UCCUAGAAUCAA 235CCCUUUAUUGAUU 233 AL-DP- 98 93 92 84 97 UAAAGGGdTdT CUAGGAdTdT 2003 6159 CUAGAAUCAAUA 237 UGCCCUUUAUUGA 240 AL-DP- 3 AAGGGCAdTdT UUCUAGdTdT2004 322 320 ACAUUUGAUAAC 239 CUUCAUUGUUAUC 242 AL-DP- 7 AAUGAAGdTdTAAAUGUdTdT 2005 323 321 CAUUUGAUAACA 241 UCUUCAUUGUUAU AL-DP- 5AUGAAGAdTdT CAAAUGdTdT 2006 324 322 AUUUGAUAACAA 243 UUCUUCAUUGUUA 244AL-DP- 4 UGAAGAAdTdT UCAAAUdTdT 2007 325 323 UUUGAUAACAAU 245CUUCUUCAUUGUU 246 AL-DP- 7 GAAGAAGdTdT AUCAAAdTdT 2008 426 424AAGUGAAAUACU 247 CAUUCCUAGUAUU 248 AL-DP- 2 AGGAAUGdTdT UCACUUdTdT 2009427 425 AGUGAAAUACUA 249 GCAUUCCUAGUAU 250 AL-DP- 7 GGAAUGCdTdTUUCACUdTdT 2010 428 426 GUGAAAUACUAG 251 AGCAUUCCUAGUA 252 AL-DP- 4GAAUGCUdTdT UUUCACdTdT 2011 429 427 UGAAAUACUAGG 253 AAGCAUUCCUAGU 254AL-DP- 96 77 68 66 92 AAUGCUUdTdT AUUUCAdTdT 2012 430 428 GAAAUACUAGGA255 GAAGCAUUCCUAG 256 AL-DP- 98 85 76 75 89 AUGCUUCdTdT UAUUUCdTdT 2013431 429 AAAUACUAGGAA 257 UGAAGCAUUCCUA 258 AL-DP- 98 85 81 68 66UGCUUCAdTdT GUAUUUdTdT 2014 550 548 GAAGCAUUAAUG 259 CAUUGGUCAUUAA 260AL-DP- 7 ACCAAUGdTdT UGCUUCdTdT 2015 551 549 AAGCAUUAAUGA 261UCAUUGGUCAUUA 262 AL-DP- 98 88 82 75 94 CCAAUGAdTdT AUGCUUdTdT 2016CGAUAAUAUAAC 263 UCUUGCUGUUAUA 264 AL-DP- 90 AGCAAGAdTsdT UUAUCGdTsdT1729 CGAUUAUAUUAC 265 UCAUCCUGUAAUA 266 AL-DP- AGGAUGAdTsdT UAAUCGdTsdT1730

TABLE 1c RSV N gene % % % % % inhibi- inhibi- inhibi- inhibi- inhibi-tion tion tion tion Actual SEQ ID SEQ ID tion RSV A2 RSV A2 RSV A2 RSV Bstart Sense  NO Antisense  NO AL-DP # (5 nM) 500 pM 50 pM 5 pM (5 nM) 3GGCUCUUAGCAAAGUC 267 CUUGACUUUGCUAA 268 AL-DP- 98 86 84 80 93 AAGdTdTGAGCCdTdT 2017 5 CUCUUAGCAAAGUCAA 269 AACUUGACUUUGCU 270 AL-DP- 2GUUdTdT AAGAGdTdT 2018 52 CUGUCAUCCAGCAAAU 271 UGUAUUUGCUGGAU 272 AL-DP-5 ACAdTdT GACAGdTdT 2019 53 UGUCAUCCAGCAAAUA 273 GUGUAUUUGCUGGA 274AL-DP- 2 CACdTdT UGACAdTdT 2020 191 UAAUAGGUAUGUUAUA 275 GCAUAUAACAUACC276 AL-DP- 3 UGCdTdT UAUUAdTdT 2021 379 AUUGAGAUAGAAUCUA 277UUCUAGAUUCUAUC 278 AL-DP- 98 78 77 75 94 GAAdTdT UCAAUdTdT 2022 897AUUCUACCAUAUAUUG 279 GUUCAAUAUAUGGU 280 AL-DP- 1 AACdTdT AGAAUdTdT 2023898 UUCUACCAUAUAUUGA 281 UGUUCAAUAUAUGG 282 AL-DP- 7 ACAdTdT UAGAAdTdT2024 899 UCUACCAUAUAUUGAA 283 UUGUUCAAUAUAUG 284 AL-DP- 96 89 84 77 96CAAdTdT GUAGAdTdT 2025

1. A device for generating an aerosol suspension of respirable particlescomprising an siRNA, wherein the device is a nebulizer or a solidparticulate therapeutic aerosol generator, and wherein the devicecomprises a composition comprising the siRNA consisting of a sensestrand and an antisense strand, wherein the sense strand consists of thenucleotide sequence of SEQ ID NO:267 and the antisense strand consistsof the nucleotide sequence of SEQ ID NO:268.
 2. The device of claim 1,wherein the device is a pressure driven jet nebulizer or an ultrasonicnebulizer.
 3. The device of claim 2, wherein the device is a pressuredriven jet nebulizer.
 4. The device of claim 1, wherein the device is asolid particulate therapeutic aerosol generator.
 5. The device of claim4, wherein the solid particulate therapeutic aerosol generator is aninsufflator.
 6. The device of claim 4, wherein the solid particulatetherapeutic aerosol generator is a metered dose inhalator.
 7. The deviceof claim 1, wherein the composition comprises a liquid carrier.
 8. Thedevice of claim 7, wherein the liquid carrier comprises a hypertonicsaline solution.
 9. The device of claim 7, wherein the liquid carriercomprises an isotonic solution.
 10. The device of claim 7, wherein thesiRNA comprises up to 40% w/w of the composition.
 11. The device ofclaim 7, wherein the siRNA comprises less than 20% w/w of thecomposition.
 12. The device of claim 1, wherein the compositioncomprises a powder.
 13. The device of claim 12, wherein the powder iscontained in capsules or cartridges.
 14. The device of claim 12, whereinthe powder comprises a powder diluent.
 15. The device of claim 1, 3, 6,9, or 14, wherein the composition comprises a therapeutic agent inaddition to the siRNA.
 16. The device of claim 1, 3, 6, 9, or 14,wherein the composition comprises an siRNA stabilizing agent selectedfrom the group consisting of a chelator and an RNAse inhibitor.
 17. Thedevice of claim 1, 3, 6, 9, or 14, wherein the device generatesrespirable particles less than 5 microns in size.
 18. A method ofreducing the levels of a Respiratory Syncytical virus (RSV) protein,mRNA, or titer in lung or respiratory cells in a subject comprising thesteps of using the device of claim 1, 3, 6, 9, or 14 on the subject. 19.A method of reducing the levels of a Respiratory Syncytical virus (RSV)protein, mRNA, or titer in lung or respiratory cells in a subjectcomprising the steps of using the device of claim 15 on the subject. 20.A method of reducing the levels of a Respiratory Syncytical virus (RSV)protein, mRNA, or titer in lung or respiratory cells in a subjectcomprising the steps of using the device of claim 16 on the subject. 21.The device of claim 1, wherein the device is a pressure driven jetnebulizer, the composition comprises an isotonic solution, the siRNAcomprises up to 40% w/w of the composition, and the device generatesrespirable particles less than 5 microns in size.
 22. The device ofclaim 21, wherein the composition comprises a therapeutic agent inaddition to the siRNA.
 23. The device of claim 21, wherein thecomposition comprises an siRNA stabilizing agent selected from the groupconsisting of a chelator and an RNAse inhibitor.
 24. A method ofreducing the levels of a Respiratory Syncytical virus (RSV) protein,mRNA, or titer in lung or respiratory cells in a subject comprising thesteps of using the device of claim 21, 22, or 23 on the subject.